PROJECT SUMMARY Fanconi anemia (FA) is a recessive disorder caused by deficient DNA damage repair. FA patients exhibit aplastic anemia, congenital abnormalities, and profoundly elevated cancer occurrence. Cells derived from FA patients are hypersensitivity to DNA crosslinking agents and highly susceptible to chromosome breakage under genotoxic stress. To date, 17 autosomal and 1 X-linked genes are designated as causative genes for FA, but the molecular structure of the FA pathway remains largely unclear. Lack of defined genetic model systems and a scarcity of recognizable protein domains in most FA proteins are among the major obstacles impeding the advance of FA biology. Recent genetic studies in nice revealed an intriguing link between the FA pathways and aldehyde metabolism and implicated DNA-protein crosslinks (DPCs) as a physiologically relevant endogenous lesion. Many DNA interstrand crosslinking (ICL) agents, such as aldehydes and cisplatin, also induce DPCs. Ionizing radiation and UV exposure also generate abundant nuclear DPCs. Therefore, DPCs is a significant type of DNA damage. Given that DPCs and ICLs are both strong obstacles of DNA transactions and that a cohort of mammalian DNA repair mutants exhibit shared ICL and DPC sensitivities, it is likely that repair of these two types of lesions assumes similar molecular mechanisms in utilizing lesion bypass synthesis, nucleotide excision repair, and FA pathway components. The main objective of Project 3 is to study how cells repair DPCs via a combined proteolytic and nucleolytic mechanisms and to determine the link between FA pathway function and endogenous DPCs potentially arisen from gene transcription reprogramming. These objectives will be achieved with two specific aims: (1) Define factors and pathways involved in DPC repair and (2) Define FA pathway function in countering endogenous DNA-crosslinking lesions. Elucidation of the DPC repair mechanism will address a critical knowledge gap in DNA repair biology. It may also reveal the underlying mechanism of the hematopoietic manifestation of FA patients. The FA pathway functions primarily in resolving replication fork-blocking DNA lesions. This type of lesion is exemplified by DNA crosslinks most frequently generated by bifunctional alkylating chemotherapeutic modalities, such as cisplatin and melphalan, and by DNA-protein crosslinks produced with high frequency from ionizing radiation exposure. For example, clinical response of many ovarian cancers to cisplatin treatment is dictated by their FA pathway status. In summary, this project is aimed at delineating the molecular pathological mechanism of Fanconi anemia with the immediate benefit of uncovering novel therapeutic targets to improve cancer treatment outcomes.